DESCRIPTION (From applicant's abstract): Conjugation of ubiquitin to Lys residues of cellular proteins is a covalent signal for degradation by the 26S protease. Ubiquitin is recycled during the degradative process by cleavage of the ubiquitin-substrate (or ubiquitin-ubiquitin) isopeptide bond(s). A surprisingly large family of deubiquitinating enzymes (isopeptidases) is now known to exist, including 17 members in S. cerevisiae. Although it is clear that some functional overlap exists among certain members of this family, it is evident that others carry out relatively selective functions. This proposal focuses mainly on two enzymes of the latter type, Doa4 and UBP14(iso T). These enzymes are proposed to play distinct, sequential roles in the final stages of 26S protease-mediated degradation. A combined biochemical and genetic approach will be taken to test specific hypotheses about the structure and function of each enzyme. The results will provide insight into the mechanism of degradation of ubiquitin conjugates, and possibly into the biological rationale for the existence of many deubiquitinating enzymes.